A Mechanism Underlies The Hepatotoxicity Of Anti-tuberculosis Drugs Mediated By Pathway Of Fxr-bile Acid Based On Targeted Metabolomics

Objective:Through studying the changes in bile acid profiles induced by anti-tuberculosis drugs in mice,the possible mechanism of FXR-bile acids mediated anti-tuberculosis drugs hepatotoxicity was explored,and to provide basis for the rational use of anti-tuberculosis drugs and the prevention of liver injury.Methods:1.Targeted metabolomics:A LC-MS/MS method for the determination of 19 bile acid concentrations in mouse serum was established,using a Shimadzu LC-30A liquid chromatography system tandem AB SCIEX QUAD 5500 mass spectrometry and CORTECS UPLC(?)HSS T3(2.1×100 mm,1.6μm)column.A gradient elution was performed using water with 0.1%formic acid and 6mM ammonium acetate(A)and 95%acetonitrile(B)as mobile phase.Electrospray ionization source(ESI)and multiple reaction monitor(MRM)mode were used to profile bile acids in serum samples;2.Animal groups with liver injury and chemical intervention:wide-type mice(C57BL/6 mice)were randomly divided into 5 groups(n=6):control(0.5%CMC-Na),Rifampin(RIF,120 mg·kg-1),Isoniazid(INH,60 mg·kg-1),Pyrazinamide(PZA,300 mg·kg-1),HRZ(120 mg·kg-1 RIF+60 mg·kg-1 INH+300 mg·kg-1 PZA),serum and liver of mice were collected.FXR knockout mice(FXR-/-)and obeticholic acid(OCA)intervention were used to observe changes in metabolites and tissue damage after treated HRZ or HRZ+OCA in FXR-/-mice and C57BL/6 mice(n=6);3.Pharmacodynamic indicators:liver function were assessed using biochemical tests for the levels of ALT,AST,DBIL,TBIL and TBA;pathological changes of hepatic tissue were assessed using HE staining;4.Gene and protein expression:RT-qPCR was employed to detect the expression of FXR/SHP/CYP7A1 genes in mouse liver and the expression of FXR protein were detected by western blot.Results:1.A LC-MS/MS method for the determination of 19 bile acid concentrations in mouse serum was established.The linear range 4-4000ng·mL-1 used for high concentration bile acids and 0.4-400ng·mL-1 used for low concentration bile acids were good,and all RSD values of intra/inter-batch precision were less than 15%.The method meeted the sample testing requirements due to its stable performance when placed in room temperature for 6h,placed in sample for 24h and repeated freezing/thawing three times at-80℃;2.After administration,liver tissue showed different degrees of liver damage in each administration group;ALT and AST were significantly increased in the INH,RIF,and HRZ groups(P<0.05),but slightly increased in the PZA group;TBA was most significantly increased in the RIF group(P<0.01);changes in 8 bile acids were mostly different,CA was significantly increased(P<0.05)and had a certain correlation with ALT and AST in C57BL/6 mice and human treated with HRZ;3.Compared with C57BL/6 mice,the increase in ALT,AST and bile acids was reduced in FXR-/-mice.The expression of FXR and SHP were decreased significantly in HRZ group,while the expression of CYP7A1 was increased.Compared with HRZ group,ALT,AST and bile acids tended to normal levels when treated with OCA,and the expression of FXR,SHP and CYP7A1 were partially reversed.Conclusions:1.The hepatotoxicity induced by anti-tuberculosis drugs was related to the primary bile acid biosynthetic pathway,CA was significantly increased and had a certain correlation with ALT and AST;2.FXR-Bile acid pathway is one of the possible mechanisms of hepatotoxicity induced by anti-tuberculosis drugs,OCA could used to partially reverse the hepatotoxicity caused by anti-tuberculosis drugs.